KR20190070317A - Compositions and Methods for Cancer Immunotherapy - Google Patents

Abstract

The present invention provides compositions and methods for combination therapy comprising administering drug-resistant immunotherapy, an immunocompromise inhibitor, and chemotherapy to a patient in need thereof for the treatment of cancer.

Description

Compositions and Methods for Cancer Immunotherapy

Related application

This application claims the benefit of U.S. Provisional Patent Application No. 62 / 376,680, filed August 18, The entire teachings of the above application (s) are incorporated herein by reference.

BACKGROUND OF THE INVENTION

 Significant progress has been made in the field of cancer detection and treatment, but treatment of terminal and metastatic cancers remains a major challenge. Cytotoxic chemotherapeutic agents remain the most widely used and successfully used anticancer drugs. However, they are not uniformly effective and the introduction of such agents together with new therapies such as immunotherapy is problematic. For example, chemotherapeutic agents can be detrimental to the establishment of potent anti-tumor immunocompetent cells due to the nonspecific toxicity profile of the agent. Small molecule-based therapies targeting cell proliferation pathways may also interfere with the formation of anti-tumor immunity. Additional complications found in the immunotherapeutic approach include down-regulation of MHC-class I antigen expression, presentation of immune checkpoint molecules on tumor or effector cell surfaces, or dissociation of soluble tumor ligands into plasma, which results in down regulation of effector cell receptors Is the ability of tumor cells to overcome the immune response of the body through. Such a process produces normally visible tumors and avoids attacks by the immune system. However, if a temporary effective chemotherapeutic regimen can be combined with both the new immunocompetent cell therapy and the immune checkpoint inhibitor, significant improvements in anti-neoplastic therapy can be achieved.

Several drug resistance genes have been identified that could potentially be used to confer drug resistance to targeted immune cells and the development of gene therapy techniques has enabled us to test the possibility of using these genes in drug resistance gene therapy research. For example, the shRNA strategy was used to reduce the level of hypoxanthine-guanine phosphoribosyltransferase (HPRT) that confers resistance to 6-thioxanine. In addition, the drug resistance gene MGMT encoding human alkyl guanine transferase (hAGT) is a DNA repair protein that confers resistance to cytotoxic effects of alkylating agents such as nitroso urea and temozolomide (TMZ). 6-Benzylguanine (6-BG) is an inhibitor of AGT that enhances nitroso urea toxicity, which is co-administered with TMZ to enhance the cytotoxic effect of this agent. Several mutant forms of MGMT that encode variants of AGT are highly resistant to 6-BG inactivation, but retain their ability to repair DNA. P140KMGMT-based drug resistance gene therapy has been shown to provide chemical protection to mice, dogs, rhesus monkeys, and human cells, particularly hematopoietic cells.

Tumor cells often express cell-surface molecules that reveal their cancerous nature. However, these same cells can also present immune checkpoint molecules on their surface that mimic normal cells, thereby avoiding immune attacks. Immune checkpoints are often regulated by interactions between specific receptor / ligand pairs, including CTLA-4 and PD-1. CTLA-4, PD-1 and its ligands are members of the CD28-B7 family of co-signaling molecules that play an important role throughout all stages of T-cell function and other cellular functions. PD-1 receptors are expressed on the surface of activated T cells (and B cells), and under normal circumstances, their ligands (PD-L1 and < RTI ID = 0.0 > PD-L2. This interaction sends the signal to the T cell, essentially blocking it or suppressing it. Cancer cells use this system by inducing high levels of PD-L1 expression on their surface. This allows them to control the PD-1 pathway and inhibit the anti-cancer immune response by blocking T-cells expressing PD-1 that can enter the tumor microenvironment.

Checkpoint inhibitors provide a means for screening tumor cells by blocking receptor / ligand pairs such as CTLA-4 or PD-1, thereby allowing T cells to initiate an immune response. Six immuno checkpoint inhibitors approved by the US Food and Drug Administration for accelerated cancer are ipilimumab (YERVOY®), pembrolizumab (KEYTRUDA®), atezolizumab (TECENTRIQ®) , Durvalumab (IMFINZI ™), avelumab (BAVENCIO), and nivolumab (OPDIVO®). YERVOY® is a monoclonal antibody that targets CTLA-4 on the surface of T cells and has been approved for treatment of melanoma. KEYTRUDA® is used to target PD-L1 and treat melanoma and non-small cell lung cancer. OPDIVO also targets PD-1 and has been approved for the treatment of melanoma, renal cell carcinoma, and non-small cell lung cancer. Additional checkpoint targets that can be proven effective include TIM-3, LAG-3, various B-7 ligands, CHK1 and CHK2 kinases, BTLA, A2aR, and the like.

Typically, activity and high objective response rates (ORR) associated with checkpoint inhibitor therapy are associated with tumors with high mutation loads. Mutation frequencies are generally associated with increased neoplastic antigen signaling leading to increased tumor immunity.

In May 2017, the US Food and Drug Administration (FDA) approved a new, non-resectable, metastatic or metastatic, microsatellite instability-high (MSI-H) or mismatch repair Accelerated approval for pembrolizumab in adult and pediatric patients with deficient solid tumors without satisfactory alternative treatment options or with MSI-H or dMMR colorectal cancer advanced after treatment with fluoropyrimidine, oxaliplatin, and irinotecan . This is the FDA's initial approval for indications, regardless of tissue / site. Patients with dMMR have defects that are incapable of correcting genetic mutations leading to hypermutated conditions.

Clinical data demonstrate that ORR increases to 62% in patients with dMMR due to their high mutation burden in cancers such as colorectal cancer, where ORR is typically 0%, with checkpoint inhibitor therapy (Le DT, et al ASCO 2015. Abstract LBA100). Similarly, analysis by Hodges et al. (Neuro-Oncology, 19 (8), 1047-1057, 2017) demonstrates that glioblastomas (GBM) typically have a low mutational burden and only 3.5% High tumor mutation load. However, in patients with GBM with dMMR, they were found to have the highest mutation load compared to other high-malignant tumors (JCO, 2016 34:19, 2206-2211). Boufett et al. Describe the remarkable and sustained response of two pediatric patients with recurrent multifocal GBM who were refractory to the current standard of care when treated with single-agent nobilurip. The dMMR mutation allowed these two patients to respond to checkpoint suppression, but their failure to respond to standard therapy could also be due to dMMR. This generally supports data suggesting that GBM is not significantly immunogenic and will not show a significant ORR upon treatment with a checkpoint inhibitor.

Since 2005, the standard-management therapy for Group 1 GBM is the Stupp protocol, which includes treatment with chemotherapy temozolomide (TMZ) (Stupp et al., N Engl J Med 2005; 352: 987-996 ). TMZ is an alkylating agent that causes double stranded DNA damage. TMZ is Miss pairing in DNA O ⁶ lead to the formation of point mutations to the AT from (mispairing) and GC - add methyl guanine (O ⁶ MeG) function thereby generating water (Roos et al, Cancer Letters 332 (2013). 237). Tolerance mechanism for the effective treatment to TMZ is O-methyl addition to removing water from the O ⁶ position ^{6-methyl-guanine-methyl} transferase (MGMT). In cells with a functional MMR system, GC to AT point mutations will cause double stranded damage after two cycles of DNA replication. As Roos notes, "O ⁶ MeG does not induce direct apoptosis; it requires MMR and DNA replication". Thus, newly diagnosed GBM patients who respond best to immunotherapy and / or checkpoint suppression due to high mutation load and neonatal burden are also the least likely to respond to conventional alkylation chemotherapy. This recognition will explain the observation that the MMR and MLH1 mutations in GBM patients actually become a negative prognosis for survival (Draaisma et al., Acta Neuropathol Commun. 2015; 3: 88). These patients do not respond to conventional treatment with TMZ.

The present invention provides a solution to this challenge. Over-mutations due to dMMR can induce more potent immunogen signaling, but they can also result in lower response to standard chemotherapeutic agents and lower survival rates. By causing double-stranded DNA damage, TMZ has demonstrated its ability to induce hypermutation and stronger immunogenic signaling in tumors. By artificially generating hypermutations with chemotherapy, the ability to maintain lymphocytes alive during chemotherapy treatment can maintain tumor sensitivity to standard therapy while increasing responsiveness to tumor immunity and checkpoint inhibition.

Unfortunately, TMZ-induced mutations over time have the potential to mutate in the MMR system, resulting in reduced sensitivity to TMZ and other alkylating agents. Without wishing to be bound by theory, hypermutations caused by TMZ or other alkylating agents are most likely to be subclonal signature 11 mutations that may not lead to long-term immunogenicity (Alexandrov et al., Nature, Vol 500, 22 Aug. 2013). However, our data suggest that in the short term, initiation of double-strand injury induces a DNA damage response that causes a transient upregulation of the NKG2D receptor ligand on the tumor cell surface and leads to the activation of innate immune responses and the activation of drug resistant γδ T- (Lamb et al., PLOS ONE, Vol 8, Issue 1, Jan. 2013).

Thus, the present invention targets both mechanisms together at the beginning of cancer progression. The DNA damage caused by TMZ, which is not bound to the theory, results in upregulation of capillary vasodilatory ataxia mutation (ATM) and capillary vasodilatory ataxia Rad-3 related (ATR) protein kinases associated with DNA damage reactions . DNA damage responses and upregulation of ATM and ATR results in the upregulation of NKG2D ligands such as MICA / B and ULBP16 on tumor cell surfaces and the initiation of anti-tumor immune responses. By upregulating the immune response through DNA damage and braking immune cells with checkpoint inhibition, and by killing most of the chemotherapy-sensitive tumors, we intend to produce a more robust, more sustained response to cancer patients .

Thus, there is an urgent need for a combination therapy that enhances, replaces or supplements the current methods of treating cancer, especially cancer, which is transiently responsive to cancer and chemotherapy. Combination therapies, including drug-resistant immune competent cells, immune checkpoint inhibitors, and chemotherapy, provide such a complementary approach.

Summary of the Invention

The present invention provides compositions and methods for enhancing the immune response of immunocompetent cells to cancer, including protection from drug-induced toxicity during chemotherapy, thereby providing a combination therapy for treating cancer, Quot; drug-resistant immunotherapy "with one or more cycles and / or doses of an inhibitor. Combinations of checkpoint inhibitors and drug resistant immunotherapy may be administered sequentially, or substantially concurrently, in any order. Drug tolerance - immunotherapy involves genetic modification of isolated cytotoxic immune cells. Preferably, the isolated cytotoxic immune cells are genetically modified using any method known in the art. Preferably, the transgenic methods include, but are not limited to, HIV-based lentiviral systems or gene editing systems. Preferably, the gene editing system comprises a zinc finger nuclease (ZFN), a transcriptional activator-like effector nuclease (TALEN), or a clustered open reading frame having regular intervals to deliver the drug tolerance-imparting genetic elements to the immunocompetent cell line Including but not limited to proteins such as Cas9 associated with structural repeats (CRISPR). Genetically engineered immunocompetent cells produce significant resistance to certain chemotherapeutic cytotoxic agents compared to unmodified cells; Such drug resistance does not affect the ability of genetically engineered cells to kill target cancer cells in the presence or absence of a chemotherapeutic agent. Such drug resistant immunotherapies are described, for example, in Spencer HT et al . in US 2015/0017137.

The present invention provides a method of treating a cell, comprising: obtaining a selectively enriched and / or selectively propagated population of cytotoxic immune cells, said cell comprising genetically modified cells such that said cytotoxic immune cells are resistant to the therapeutic; Administering to a patient in need thereof an effective amount of a therapeutic agent that is resistant to said genetically engineered cells to a patient in need thereof with a population of genetically modified cytotoxic immune cells and further administering to said patient an effective amount of at least one immune checkpoint inhibitor, A method of treating cancer in a patient, comprising the step of treating a cancer in a patient.

Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises gamma delta T-cells. Preferably, the population of cytotoxic immune cells is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% Of γδ T-cells. Preferably, the population of cytotoxic immune cells comprises about 50% to about 95% of the gamma delta T-cells. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises gamma delta T-cells and natural killer (NK) cells. Preferably, the population of cytotoxic immune cells comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% . Preferably, the population of cytotoxic immune cells comprises about 5% to about 25% of NK cells. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the present invention includes gamma delta T-cells, NK cells and other immunologically competent cells, including but not limited to monocytes, dendritic cells and macrophages. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the present invention comprises gamma delta T-cells derived from human induced pluripotent stem cells (hiPSC).

Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises NK cells. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises NK cells and gamma delta T-cells. Preferably, the population of cytotoxic immune cells comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% . Preferably, the population of cytotoxic immune cells comprises about 5% to about 25% of NK cells. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention includes NK cells, gamma delta T-cells, and other immunocompetent cells including, but not limited to, monocytes, dendritic cells, and macrophages. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises NK cells derived from human induced pluripotent stem cells (hiPSC).

Preferably, the step of obtaining a population of cytotoxic immune cells that are genetically modified to be resistant to the therapeutic agent can be accomplished by, for example, obtaining a biological sample, including, but not limited to, a tissue sample comprising blood or a tumor biopsy from the subject , To obtain a population of cytotoxic immune cells from a subject such as a human or animal subject. The sample may be selectively enriched for cytotoxic immune cells and other immunocompetent cells and / or cells present in the sample may be selectively propagated to increase the population of cells present in the sample. The cytotoxic immune cells are preferably stably transfected or genetically edited with a vector comprising a heterologous nucleic acid sequence operably linked to a promoter, wherein said heterologous nucleic acid sequence is a polypeptide that confers cell resistance to one or more chemotherapeutic agents Lt; / RTI >

Preferably, the present invention encompasses a cytotoxic therapeutic agent having the property of suppressing the survival of cancer cells, gamma delta T-cells, NK cells, or any combination thereof, optionally further comprising other immunologically competent cells, A population of cytotoxic immune cells that are genetically modified to be resistant to the therapeutic agent, and an immune checkpoint inhibitor that blocks cell-surface proteins on cancer cells.

Preferably, the present invention relates to a therapeutic agent having the property of inhibiting the survival of cancer cells and inducing stress proteins in cancer cells, a population of cytotoxic immune cells, and an immune checkpoint inhibitor blocking cell-surface proteins on cancer cells A cytotoxic immune cell comprising a gamma delta T-cell, an NK cell, or any combination thereof, and optionally further immunologically competes with another immunocompetent cell, And the cytotoxic immune cells are genetically modified to be resistant to the therapeutic agent.

Preferably, the population of cytotoxic immune cells used in the compositions and methods of the present invention is gamma delta T-cells, wherein about 5%, 10%, 20%, 30%, 40%, 50% , 60%, 70%, 80%, 90%, or more than 95% express a polypeptide conferring resistance to a chemotherapeutic agent), or a separate composition comprising γδ T-cells, And more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the nucleic acid comprises a nucleic acid encoding a polypeptide conferring resistance to a chemotherapeutic agent ), Or a γδ T-cell comprising a nucleic acid encoding a polypeptide conferring resistance to a chemotherapeutic agent. Preferably, a polypeptide that imparts resistance to the chemotherapeutic agents is O ⁶ methyl guanine DNA methyl transferase (MGMT), dihydro folate drug-resistant variants (L22Y-DHFR) of the reductase, thymidine delay tree synthetase, and / Or multidrug resistance-1 protein (MDR1).

Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises NK cells, wherein about 5%, 10%, 20%, 30%, 40%, 50% 60%, 70%, 80%, 90%, or more than 95% express polypeptides conferring resistance to chemotherapeutic agents. Preferably, the cytotoxic immune cells used in the compositions and methods of the present invention comprise NK cells, wherein greater than about 50% of the NK cells express a polypeptide conferring resistance to the chemotherapeutic agent. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention is about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90%, or more than 95% of the NK cells encode polypeptides conferring resistance to chemotherapeutic agents. Preferably, the cytotoxic immune cells used in the compositions and methods of the invention comprise NK cells comprising a nucleic acid encoding a polypeptide wherein greater than about 50% of the NK cells confer resistance to the chemotherapeutic agent. Preferably, the polypeptide is expressed by NK cells, which confer resistance to chemotherapeutic agents is O ⁶ methyl guanine DNA methyl transferase (MGMT), dihydro folate drug-resistant variants (L22Y-DHFR) of the reductase, thymidine delay Synaptase, and / or multidrug resistance-1 protein (MDR1).

Preferably, the present invention provides a composition comprising at least one checkpoint inhibitor, and a separate population of cytotoxic immune cells comprising a gamma delta T-cell, an NK cell, or any combination thereof, wherein the cell More than about 5%, and preferably greater than about 50%, of the population of toxic immune cells express a polypeptide that confers resistance to a therapeutic agent capable of inhibiting cancer cell survival.

These and other objects, features and advantages of the present invention will become apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings, in which like reference numerals refer to like parts . The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
Figure 1 shows checkpoint molecule expression for < RTI ID = 0.0 > y8 < / RTI > T cells prior to in vitro culture of PBMCs from Donor 1.
Figure 2 shows the up-regulation of checkpoint molecules for in vitro enriched < RTI ID = 0.0 > [gamma] < / RTI > T cells from donor 1.
Figure 3 shows the checkpoint molecule expression for < RTI ID = 0.0 > y8 < / RTI > T cells prior to in vitro culture of PBMCs from Donor 2.
Figure 4 shows up-regulation of checkpoint molecules for in vitro enriched < RTI ID = 0.0 > [gamma] < / RTI > T cells from Donor 2.
Figure 5 shows upregulation of PD-1 and CTLA-4 on human gamma delta T cells upon stimulation with IL2 and Zol.
Figure 6 shows PD-L1 expression on glioblastoma tumor cells.
Figure 7 shows the cytotoxicity of GMP produced γδ T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is not limited to the specific embodiments described, but of course they can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as the scope of the invention is to be limited only by the appended claims.

Where a range of values is provided, each intervening value up to one tenth of the lower limit unit between the upper and lower limits of the range (unless explicitly stated otherwise in context) Other stated or intervened values of " a " are understood to be encompassed within the present invention. The upper and lower limits of such smaller ranges can be included independently in smaller ranges and are also encompassed within the invention, subject to any limitations specifically excluded from the ranges mentioned. Where the stated range includes the limit of one or both, ranges excluding one or both of the included limits are also included in the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and disclose the method and / Incorporated herein by reference in its entirety. The citation of any publication is for that invention prior to the filing date and should not be construed as acknowledging that the invention is not entitled to precede such publication due to prior art inventions. In addition, the date of the publication provided may be different from the actual publication date, which may need to be verified independently.

Unless otherwise indicated, the description describes techniques such as chemistry, synthetic organic chemistry, biochemistry, biology, molecular biology, molecular imaging, etc. within the skill of the art. Such techniques are fully described by the literature.

The following examples are presented to provide those of ordinary skill in the art with a complete invention and description of how to carry out the method and how to use the compositions and compounds disclosed and claimed herein.

Unless otherwise indicated, the invention is not limited to specific materials, reagents, reactants, manufacturing processes, and the like, which may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular functions only, and is not intended to be limiting. It is also possible in the present invention that the steps can be performed in a logically different possible order.

It should be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "support" includes a plurality of supports. In the present specification and the following claims, unless the intent is explicitly contemplated, a number of terms will be mentioned which are defined to have the following meanings.

Justice

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions given below.

"Administration" means introducing a compound of the invention, a biological material comprising a population of cells, or a combination thereof, into a human or animal subject. One preferred route of administration of the compounds is intravenous. Other preferred routes of administration of the compounds can be administered intraperitoneally or intraperitoneally, or via the catheter to the brain. However, any route of administration may be used, such as oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, cerebrospinal fluid, or drip infusions into body compartments. Direct injection into a target tissue site, such as a solid tumor, is also contemplated.

The term "therapeutic agent" as used herein refers to a compound or derivative thereof capable of interacting with a cancer cell, reducing the proliferative state of the cell and / or killing the cell. Examples of therapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, iospamide); Metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or derivatives thereof); Substituted nucleotides; Substituted nucleosides; DNA demethylating agents (also known as antimetabolites; for example, azacitidine); Antitumor antibiotics (e. G., Mitomycin, adriamycin); Plant-derived antineoplastic agents (e. G. Vincristine, vindesine, TAXOL, paclitaxel, abraxane); Cisplatin; Carboplatin; But are not limited to, chemotherapeutic agents including, but not limited to, etoposide, and the like. Such agents include the anticancer agents trimethotrexate (TMTX); Temozolomide; Raltitrexed; S- (4-nitrobenzyl) -6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); (BCNU; carmustine), lomustine (CCNU) +/- Procarbazine, and Vincristine (PCV, for example) E. G., Fotemustine); Cytarabine; And camptothecin; Or any therapeutic derivatives thereof. ≪ Desc / Clms Page number 7 >

The term "therapeutically effective amount " as used herein refers to that amount of a compound or therapeutically active composition to be administered which moderates to some extent one or more of the symptoms of the disease, disorder, or disorder being treated. In relation to cancer or pathology associated with uncontrolled cell division, a therapeutically effective amount may be determined by (1) reducing the size of the tumor and / or (2) inhibiting abnormal cell division, e.g., cancer cell division, and / , (3) prevent or reduce the metastasis of cancer cells, and / or (4) inhibit or prevent metastasis of cancer cells that are associated with or are partly caused by uncontrolled or abnormal cell division, for example, Refers to an amount that has an effect of mitigating (or preferably eliminating) some degree of pathology, including cancer, or one or more symptoms associated with angiogenesis.

The term "treating" or "treatment" of a disease (or disorder or disorder) as used herein is intended to prevent the disease from occurring in a human or animal subject that is susceptible to disease but has not yet experienced or exhibited symptoms of the disease Refers to inhibiting (retarding or inhibiting the development) of a disease, providing relief from symptoms or side effects of the disease (including palliative treatment), and causing regression of the disease. With respect to cancer, this term also means that the average life span of an individual with cancer may increase or that one or more of the symptoms of the disease will be reduced. In the context of cancer, "treating" also includes enhancing or prolonging anti-tumor response in a subject.

As used herein, any of the dosage forms of "combination", "combination therapy" and / or "combined therapy" may be administered simultaneously, separately or in combination, or at different times a few minutes, Refers to at least two therapeutically active drugs or compositions that can be administered sequentially, but which work together in any manner to provide the desired therapeutic response.

As used herein, the term "enhancement" means that a subject or tumor cell will have improved ability to respond to the treatment disclosed herein. For example, the enhanced response may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 75%, 80%, 85%, 90%, 95% or 98% or more. As used herein, an "enhancement" may also refer to increasing the number of subjects responding to treatment, such as a combination therapy comprising chemotherapy, drug-resistant immunocompetent cells, and an immuno checkpoint inhibitor. For example, an improved response may refer to the total percentage of subjects responding to treatment, and the percentage may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35% %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more.

As used herein, the terms "subject" and "patient" include humans, mammals (eg, cats, dogs, horses, etc.), living cells, and other living organisms. A living organism can be as simple as a single eukaryotic cell, for example, or as complex as a mammal. A typical patient is a mammal, particularly a primate, especially a human. In veterinary applications, livestock such as cattle, sheep, goats, cattle, pigs, etc .; Poultry, for example, chickens, ducks, geese, turkeys and the like; And a wide variety of subjects such as domesticated animals, especially pets, such as dogs and cats, will be suitable. For diagnostic or research applications, a wide variety of mammals, including rodents (e.g., mice, rats, hamsters), rabbits, primates, Preferably, the system comprises a sample and a subject. The term "living host" refers to the above-mentioned host or organism that is alive and dead. The term "living host" refers not only to the whole host or organism, but also to the site of abstinence (e.g. liver or other organ) from the living host.

The term " gamma delta T-cells " (gamma delta T-cells) as used herein refers to a small subset of T-cells expressing distinct T-cell receptors (TCRs) on their surface. Most T-cells have a TCR consisting of two glycoprotein chains called the a- and beta-TCR chains. In contrast, in γδ T-cells, the TCR consists of one γ-chain and one δ-chain. These groups of T-cells are usually found to be the most abundant in the intestinal mucosa, within the population of lymphocytes known as intra-epithelial lymphocytes (IEL), much less common than the alpha beta T-cells. Antigenic molecules that activate γδ T-cells are not yet known. However, γδ T-cells are unusual in that they appear to not require MHC presentation and antigen treatment of peptide epitopes, even though some recognize MHC class IB molecules. Moreover, gamma delta T-cells play an important role in the recognition of lipid antigens and are believed to respond to stress-related antigens such as MIC-A and MIC-B.

As used herein, the term "human induced pluripotent stem cells" (hiPSC) refers to stem cells that are re-introduced into an embryo-like pluripotent state that enables the production of any other type of human cell, Quot; refers to the type of pluripotent stem cells that can be produced directly from human adult cells, such as programmed skin or blood cells. Human adult cells that can be obtained from patients or adult cells to be treated with hiPSC can be obtained from different individuals. Human adult cells can be transformed into pluripotent stem cells by, for example, a retroviral system or a lentiviral system, to introduce a gene encoding a transcription factor capable of converting adult cells into pluripotent stem cells .

As used herein, the term "antibody" refers to an immunoglobulin or portion thereof and includes any polypeptide comprising an antigen-binding site, regardless of source, source species, method of production, and characteristics. The antibody may be composed of a heavy chain and / or a light chain or a fragment thereof. Antibodies or antigen-binding fragments, variants or derivatives thereof of the present invention can be used in combination with a polynucleotide, monoclonal, multispecific, human, humanized, primatized, or chimeric antibody, single chain antibody, epitope- For example, an antibody comprising any one of Fab, Fab 'and F (ab') ₂ , Fd, Fvs, single-chain Fvs (scFv), single-chain antibody, disulfide-linked Fvs (sdFv), VL or VH domains Fragments, fragments produced by Fab expression libraries, and anti-idiotypic (anti-Id) antibodies. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The immunoglobulin or antibody molecule of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), species (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 And IgA2) or a subclass.

The term " biological therapeutic agent "or" biological agent "as used herein refers to any pharmaceutical agent produced or extracted from a biological source. Biopharmaceuticals are distinguished from chemically synthesized pharmaceutical products. Examples of biologic agents include recombinant therapeutic proteins, including vaccines, blood or blood components, allergens, somatic cells, gene therapy agents, tissues, antibody therapeutics and fusion proteins, and living cells. The biologic agent may be composed of a saccharide, protein or nucleic acid or a complex combination of such substances, or it may be a living entity such as cells and tissues. Biologics can be isolated from a variety of natural sources - humans, animals, or microorganisms and can be produced by biotechnology and other techniques. Specific examples of biological therapeutic agents include, but are not limited to, immunostimulants, T cell growth factors, interleukins, antibodies, fusion proteins, and vaccines such as cancer vaccines.

As used herein, the term " cancer "will be given in its ordinary sense as a generic term for a disorder in which abnormal cells are uncontrolled and divisive. In particular, and in the context of embodiments of the invention, the cancer refers to an angiogenesis-related cancer. Cancer cells can invade nearby tissues and spread to other parts of the body through the bloodstream and the lymphatic system. There are several major types of cancer, for example, carcinoma is cancer that starts in tissues that cover or cover the skin or internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissue. Leukemia is a cancer that begins in blood-forming tissues such as bone marrow and causes a number of abnormal blood cells to be produced and into the bloodstream. Lymphoma is a cancer that begins in the cells of the immune system.

When normal cells lose their ability to behave as a specific, controlled and coordinated unit, tumors are formed. In general, solid tumors are usually abnormal tissue masses that do not contain cysts or liquid areas (some brain tumors have central necrotic areas filled with cysts and liquid). A single tumor can even have a different population of cells within it and represent different processes beyond prediction. Solid tumors may be benign (not cancerous) or malignant (cancerous). Different types of solid tumors are named depending on the type of cells they form. Examples of solid tumors are sarcoma, carcinoma, and lymphoma. Leukemia (cancer of the blood) generally does not form solid tumors.

Representative cancers are, in particular, acute lymphoblastic leukemia, adult; Acute lymphoblastic leukemia, pediatric; Acute myeloid leukemia, adult; Adrenocortical carcinoma; Adrenocortical carcinoma, pediatric; AIDS-related lymphoma; AIDS-related malignant tumors; Anal cancer; Astrocytoma, pediatric cerebellum; Astrocytoma, pediatric cerebrum; Cholangiocarcinoma, liver; Bladder cancer; Bladder cancer, pediatric; Bone cancer, osteosarcoma / malignant fibrous histiocytoma; Glioblastoma, pediatric; Glioblastoma, adult; Brain stem glioma, pediatric; Brain tumor, adult; Brain tumor, brain stem glioma, pediatric; Brain tumor, cerebellar astrocytoma, pediatric; Brain tumor, brain astrocytoma / malignant glioma, pediatric; Brain tumor, ventricular tumor, pediatric; Brain tumor, sublingual bladder, pediatric; Brain tumor, suprarenal primitive neuroectodermal tumor, pediatric; Brain tumors, visual pathways and hypothalamic glioma, pediatric; Brain tumor, pediatric (other); Breast cancer; Breast cancer and pregnancy; Breast cancer, pediatric; Breast cancer, male; Bronchial adenoma / carcinoid, pediatric: carcinoid tumor, pediatric; Carcinoid tumor, upper gastrointestinal tract; Carcinoma, adrenal cortex; Carcinoma, islet cell; Primary site stomach cancer; Central nervous system lymphoma, primary; Cerebellar astrocytoma, pediatric; Brain astrocytoma / malignant glioma, pediatric; Cervical cancer; Pediatric cancer; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorder; Transparent cell sarcoma of hay; Colon cancer; Colorectal cancer, pediatric; Skin T-cell lymphoma; Endometrial cancer; Ventricular, pediatric; Epithelium, ovary; Esophagus cancer; Esophageal cancer, pediatric; Ewing family of tumors; Extracellular germ cell tumor, pediatric; Testicular germ cell tumor; Extrahepatic cholangiocarcinoma; Snow cancer, guide melanoma; Ocular cancer, retinoblastoma; Gallbladder cancer; Gastric cancer; Gastric cancer, pediatric; Gastric carcinoid tumor; Germ cell tumor, extraocular, pediatric; Germ cell tumors, testis external; Germ cell tumor, ovary; Pregnancy chorionic villi; Glioma, pediatric brain stem; Glioma, pediatric visual pathway and hypothalamus; Hair cell leukemia; Head and neck cancer; Hepatocellular carcinoma, adult (primary); Hepatocellular carcinoma, pediatric (primary); Hodgkin lymphoma, adult; Hodgkin lymphoma, pediatric; Hodgkin lymphoma during pregnancy; Hypopharynx; Hypothalamic and visual pathway glioma, pediatric; Guide melanoma; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Laryngeal cancer, pediatric; Leukemia, acute lymphoblastic, adult; Leukemia, acute lymphoblastic, pediatric; Leukemia, acute bone marrow, adult; Leukemia, acute myeloid, pediatric; Leukemia, chronic lymphocytes; Leukemia, chronic myelogenous; Leukemia, hair cells; Lips and oral cancer; Liver cancer, adult (primary); Liver cancer, pediatric (primary); Lung cancer, non-small cell; Lung cancer, small cell; Lymphocytic leukemia, adult acute; Lymphoblastic leukemia, pediatric acute; Lymphocytic leukemia, chronic; Lymphoma, AIDS-related; Lymphoma, central nervous system (primary); Lymphoma, skin T-cell; Lymphoma, Hodgkin, adult; Lymphoma, hojiquin; Infant; Lymphoma, hojiquin during pregnancy; Lymphoma, nonhoshkin, adult; Lymphoma, non-Hodgkin's, pediatric; Lymphoma, non-pregnant during pregnancy; Lymphoma, primary central nervous system; Polymer globulinemia, valdystrom; Male breast cancer; Malignant mesothelioma, adult; Malignant mesothelioma, pediatric; Malignant thymoma; Sublingual bladder, pediatric; Melanoma; Melanoma, guide; Merkel cell carcinoma; Mesothelioma, malignant; Metastatic squamous cell carcinoma with latent primary; Multiple endocrine neoplasm syndrome, pediatric; Multiple myeloma / plasma cell neoplasms; Bacterial sarcoma; Myelodysplastic syndrome; Myeloid leukemia, chronic; Bone marrow leukemia, pediatric acute; Myeloma, multiple; Myeloproliferative disorder, chronic; Nasal and sinus cancer; Biotin; Pyelonephritis, pediatric; Neuroblastoma; Neurofibroma; Non-Hodgkin's lymphoma, adult; Non-Hodgkin's lymphoma, pediatric; Non-Hodgkin's lymphoma during pregnancy; Non-small cell lung cancer; Oral cancer, pediatric; Oral cancer and lip cancer; Mouth cancer; Malignant fibrous histiocytoma of osteosarcoma / bone; Ovarian cancer, pediatric; Ovarian carcinoma; Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, pancreatic cancer of child, island cell; Sinus and nasal cancer; Parathyroid cancer; Penile cancer; Chromium-rich cell tumor; Pineal plexus and suprarenal primitive neuroectodermal tumor, pediatric; Pituitary tumor; Plasma cell neoplasm / multiple myeloma; Pleural mesothelioma; Pregnancy and breast cancer; Pregnancy and Hodgkin lymphoma; Pregnancy and non-Hodgkin's lymphoma; Primary central nervous system lymphoma; Primary liver cancer, adult; Primary liver cancer, pediatric; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Renal cell carcinoma, pediatric; Renal and ureter, transitional cell carcinoma; Retinoblastoma; Rhabdomyosarcoma, pediatric; Salivary gland cancer; Cancer of the salivary gland, pediatric; Sarcoma, Ewing family of tumors; Breeding, kaposhi; Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; Sarcoma, rhabdomyosarcoma, pediatric; Sarcoma, soft tissue, adult; Sarcoma, soft tissue, pediatric; Trestle syndrome; cutaneous cancer; Skin cancer, pediatric; Skin cancer (melanoma); Skin carcinoma, Merkel cells; Small cell lung cancer; Small bowel cancer; Soft tissue sarcoma, adult; Soft tissue sarcoma, pediatric; Metastatic, metastatic; Gastric cancer; Gastric cancer, pediatric; Primitive neuroectodermal tumor, pediatric; T-cell lymphoma, skin; Testicular cancer; Thymic gland, pediatric; Thymic gland, malignant; Thyroid cancer; Thyroid cancer, pediatric; Transitional cell carcinoma of the pyelonephritis and ureter; Trophic tumor, pregnancy; Primary stomach cancer, pediatric; Abnormal cancer of childhood; Ureters and kidneys, transitional cell carcinoma; Urethral cancer; Uterine sarcoma; Vaginal cancer; Visual pathways and hypothalamic glioma, pediatric; Vulvar cancer; Valdenstrom polymer globulinemia; And Wallace's tumors.

Tumors can be classified as malignant or benign. In both cases, there is abnormal aggregation and proliferation of the cells. In the case of malignant tumors, these cells behave more aggressively and acquire increased invasive properties. Ultimately, tumor cells will evolve out of the microscopic environment from which they originate, spread to other areas of the body (in very different environments, which generally do not help their growth) and continue rapid growth and division at this new location You can get the ability to. This is called the former. Once malignant cells have metastasized, healing is more difficult to achieve. Benign tumors tend to be less invasive and less likely to metastasize.

Brain tumors spread widely within the brain but usually do not migrate outside the brain. Glioma is very invasive across the brain, even across the hemisphere. Yet they divide in an uncontrolled way. Depending on their location, they can be life-threatening like a malignant lesion. An example of this would be benign tumors in the brain, which grow and occupy space within the skull, creating increased pressure in the brain.

As used herein, the term "enriched" refers to one or more cytotoxic immune cell types present in a sample (eg, γδ T (eg, γδ T), as compared to the total percentage of one or more of the same cell types prior to enrichment, -Cells and / or NK cells). ≪ / RTI > For example, a "concentrated" sample for one or more types of cytotoxic immune cells may contain about 10% to 100% of one or more cytotoxic immune cell types in the sample, while a cytotoxic immune The total percentage of one or more of the cell types is, for example, 0% to 10%. Preferably, the concentrated sample contains at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% Lt; RTI ID = 0.0 > immune < / RTI > The sample may be concentrated for one or more cell types using standard techniques, e. G., Flow cytometry techniques.

As used herein, the term "highly enriched" means that at least one cytotoxic immune cell type is present in at least about 70% to about 100% of a cytotoxic immune cell type in a sample, , While the total percentage of the same type of cytotoxic immune cells before enrichment is, for example, 0% to 10%. Preferably, the highly concentrated sample comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of one or more types of cytotoxic immune cells. The sample may be highly concentrated for one or more cell types using standard techniques, e. G., Flow cytometry techniques.

The term "proliferated" and "proliferation ", as used herein in connection with the proliferation of one or more cytotoxic immune cells in a sample, refers to the number of cytotoxic immune cells in one or more of the sample, for example, Quot; means increasing about 5 times, preferably at least 10 times, preferably at least about 50 times. The proliferation of a population of cytotoxic immune cells can be accomplished by any number of methods known in the art. For example, T-cells can be rapidly proliferated using nonspecific T-cell receptor stimulation in the presence of donor lymphocytes and interleukin-2 (IL-2) or interleukin-15 Is preferred. Non-specific T-cell receptor stimulation can include approximately 30 ng / ml OKT3, which is a mouse monoclonal anti-CD3 antibody (available from ORTHO-MCNEIL®, Raritan, NJ). Alternatively, the T-cells can be rapidly proliferated by stimulating peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens of cancer (including antigenic portions thereof, such as epitope (s), or cells) (HLA-A2) binding peptide < / RTI > (HLA-A2) binding peptide, optionally in the presence of a T-cell growth factor, for example, 300 IU / ml of IL-2 or IL-15, , Such as 0.3 [mu] M MART-1: 26-35 (27 L) or gp100: 209-217 (210M).

The term "fusion protein" as used herein includes, for example, an immunoglobulin antigen-binding domain having at least one target binding site, and at least one heterologous moiety, Refers to chimeric molecules. Amino acid sequences may generally be present in separate proteins taken together in a fusion polypeptide, or they may be present in the same protein, but are arranged in a new arrangement in fusion polypeptides. Fusion proteins can be generated, for example, by chemical synthesis, or by generating and translating polynucleotides in which the peptide regions are coded in the desired context.

As used herein, the term "reducing cancer" refers to a decrease in the size or volume of a tumor mass, a decrease in the number of metastasized tumors in a subject, a decrease in the proliferative state of cancer cells .

As used herein, the terms "isolated" and "isolated cell population" refer to a cell or a plurality of cells removed from a tissue or condition found in a subject. The term may further include cells isolated according to parameters such as, but not limited to, cell surface markers, reporter markers such as dyes, or labels.

The term " expressed "or" expression ", as used herein, refers to the transcription from a gene to provide an RNA nucleic acid molecule that is at least partially complementary to one of the two nucleic acid strands of the gene. The term " expressed "or" expression ", as used herein, also refers to translation from the RNA nucleic acid molecule to provide a protein, polypeptide, or portion or fragment thereof.

As used herein, the term "promoter" refers to a DNA sequence that determines the transcription initiation site from an RNA polymerase. The "promoter-proximal element" may be a regulatory sequence within about 200 base pairs of the transcription start site.

The term "recombinant cell" refers to a cell having a new combination of nucleic acid segments that are not covalently linked to each other in nature. The new combination of nucleic acid segments can be introduced into an organism using a wide variety of nucleic acid manipulation techniques available to those skilled in the art. The recombinant cell may be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell. Recombinant cells can harbor extra-genetic vectors. The extracellular nucleic acid vector is not inserted into the cell's genome. The recombinant cell may further contain a vector in the genome or a portion thereof. The term "in the genome" defines a nucleic acid construct integrated into the genome of a recombinant cell.

As used herein, the terms "recombinant nucleic acid" and "recombinant DNA" refer to combinations of at least two nucleic acid sequences that are not naturally found in eukaryotic or prokaryotic cells. Nucleic acid sequences include, but are not limited to, nucleic acid vectors, gene expression control elements, origin of replication, appropriate gene sequences that confer antibiotic resistance upon expression, protein-coding sequences, and the like. The term "recombinant polypeptide" is intended to mean a polypeptide which is produced by recombinant DNA technology and which differs from the naturally occurring polypeptide in its position, purity or structure. Generally, such recombinant polypeptides will be present in the cell in an amount different from that normally observed in nature.

The term "operably" or "operably linked ", as used herein, refers to the arrangement of coding and control sequences to perform the desired function. Thus, a control sequence operably linked to a coding sequence can perform expression of the coding sequence. Coding sequences are operably linked to a transcriptional regulatory region in a cell when the DNA polymerase binds to the promoter sequence and transcribes the coding sequence into an mRNA that can be translated into a coded protein, or under the control of a transcriptional regulatory region. The control sequences need not be contiguous as long as they function to direct the expression of the coding sequence. Thus, for example, an intervening transcribed sequence that is not yet translated may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

The terms "heterologous" and "exogenous" are intended to include sequences that are not normally associated with a region of a recombinant construct or with a particular chromosomal locus, or that are not normally associated with a particular cell when they are associated with a nucleic acid sequence such as a coding sequence and a control sequence . Thus, a "heterologous" region of a nucleic acid construct is an identifiable segment of a nucleic acid or nucleic acid attached to another nucleic acid molecule in another nucleic acid molecule not found in association with other molecules in nature. For example, the heterologous region at the end of the construct may contain a coding sequence located on the side of the sequence that is not found in nature in relation to the coding sequence. Another example of a heterologous coding sequence is a construct in which the coding sequence itself is not found in nature (e. G., A synthetic sequence having a different codon from the natural gene). Similarly, host cells transformed with constructs not normally present in the host cell will be considered heterogeneous for the purposes of the present invention.

Preferably, the promoter will be altered by addition or deletion of the sequence, or alternatively replaced with alternative sequences comprising sequences that may be natural and synthetic sequences as well as synthetic and natural sequence combinations. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter element. The electrons located upstream of the transcription initiation site are involved in inducing RNA polymerase to initiate transcription at the correct site while the latter are responsible for the transcription rate and are upstream of the TATA box. Enhancer elements can also stimulate transcription from linked promoters, but many function exclusively in certain cell types. Many enhancer / promoter elements derived from viruses, e.g., SV40, Rous sarcoma virus (RSV), and CMV promoters are active in a wide variety of cell types and are referred to as "constant" or "ubiquitous". The nucleic acid sequence inserted into the cloning site may have any open reading frame encoding the polypeptide of interest, provided that the coding sequence can block the production of the appropriate mRNA molecule and / or splice abnormally when coding the polypeptide of interest Or there should be no potential splice sites capable of producing abnormal mRNA molecules.

Since the termination region appears to be relatively interchangeable, the termination region that is primarily used will be one of convenience. The termination region may be native to the nucleic acid sequence of interest or may originate from another source.

The term "targeting agent" as used herein refers to any therapeutic molecule that targets any aspect of the immune system.

The term "vector" as used herein refers to a polynucleotide consisting of a single strand, a double strand, a circular, or a supercoiled DNA or RNA. Typical vectors may consist of the following elements operably linked at appropriate distances to allow functional gene expression: a replication origin, a promoter, an enhancer, a 5 'mRNA leader sequence, a ribosome binding site, a nucleic acid cassette, Annealing sites, and selectable marker sequences. One or more of these elements may be omitted in a particular application. The nucleic acid cassette may contain restriction sites for insertion of the expressed nucleic acid sequence. In a functional vector, the nucleic acid cassette contains an expressed nucleic acid sequence comprising translation initiation and termination sites.

The vector is constructed such that a particular coding sequence is located in a vector with the appropriate regulatory sequence, and the position and orientation of the coding sequence relative to the control sequence is such that the coding sequence is transcribed under "control" of the control or regulatory sequences. Modifications of sequences encoding a particular protein of interest may be desirable to achieve this goal. For example, in some cases, the sequence may be modified so that the sequence can be attached to the control sequence in an appropriate orientation; There may be a need to maintain the decipherment framework. Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into the expression vector, which already contains the appropriate restriction sites within the coding frame and under the control of the control sequences and control sequences.

The term " lentivirus-based vector "as used herein refers to a lentiviral vector designed to operatively insert an exogenous polynucleotide sequence into a host genome in a site-specific manner. The lentivirus-based targeting vector may, but is not limited to, be based on, for example, HIV-1, HIV-2, an immunogenic deficiency virus (SIV), or a cat immune deficiency virus (FIV). Preferably, the lentivirus-based targeting vector is an HIV-based targeting vector. This vector may comprise all or part of the polynucleotide sequence of HIV.

The terms "transformation", "transduction" and "transduction" all refer to the introduction of a polynucleotide into an acceptor cell or cells.

Combination of drug-resistant immunotherapy and immune checkpoint inhibitor

A major limitation of chemotherapy treatment for cancer is drug-induced immunotoxicity, which results in the death of immune competent cells, which otherwise would avoid undesirable infections or provide protection against cancer cells, and the loss of an effective immune system. One strategy to overcome the severe toxic effects of chemotherapy is to selectively genetically modify cytotoxic immune competent cells by introducing a retroviral vector designed to express the drug-resistant cDNA sequence, Cancer cells that can resist simultaneous administration can be actively targeted.

Immune checkpoint proteins regulate T cell function in the immune system. T cells play a central role in cell-mediated immunity. The checkpoint protein interacts with a specific ligand that essentially signals or blocks T cell function by sending a signal to the T cell. By using such a system that induces the control of T cells expressing expression checkpoint proteins on the surface of T cells entering the tumor microenvironment by inducing expression of high levels of checkpoint proteins on their surface, It inhibits the anti-cancer immune response. Thus, inhibition of checkpoint proteins will result in the recovery of T cell function and an immune response against cancer cells. Examples of checkpoint proteins include CTLA-4, PDL1 (B7-H1, CD274), PDL2 (B7-DC, CD273), PD1, B7-H3 (CD276), B7- Expression on all NK, γδ, and memory CD8 ⁺ (αβ) T cells belonging to the molecule of BTLA (CD272), HVEM, TIM3 (HAVcr2), GAL9, LAG3 (CD223), VISTA, KIR, 2B4 But are not limited to, CD160 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinase, OX40, A2aR and various B-7 family ligands.

Programmed cell death protein 1 (PD-1) is a 288 amino acid cell surface protein molecule that is expressed in T cells and pro-B cells and plays a role in their fate / differentiation. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 protein is up-regulated in macrophages and dendritic cells (DCs) in response to LPS and GM-CSF therapy, and in T and B cells in TCR and B cell receptor signaling. PD-1 negatively regulates T cell responses.

PD-1 plays a role in tumor-specific escape from immune surveillance. PD-1 has been shown to be highly expressed in tumor-specific cytotoxic T lymphocytes (CTL) in both chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML). PD-1 is also upregulated in melanoma invasive T lymphocytes (TIL) [Dotti (2009) Blood 114 (8): 1457-58]. The tumors were found to express PD-I ligands PDL-1 and PDL-2. For example, PDL-1 is found in glioblastomas, particularly in mesenchymal subtypes. (Nat Rev Neurol 2015: 11: 504-515). Expression of PDL-1 and PDL-2 by tumors in combination with up-regulation of PD-1 in CTL may be a contributing factor in the inability of CTL to mediate loss of T cell function and an effective anti-tumor response. In mice chronically infected with lymphocyte chorioamnionitis virus (LCMV), the researchers found that administration of anti-PD-1 antibodies blocked PD-1-PDL interactions and could restore some T cell function (proliferation and cytokine secretion) And decreased viral load (Barber et al (2006) Nature 439 (9): 682-687).

Tumor cells themselves express PD-L1 and are thought to limit T cell responses through this mechanism. It has further been found that inhibition of PD-I proliferates effector T cells and restricts the population of T regulatory cells in the B16 melanoma model. Blocking PD-1, CTLA-4 or IDO (indoleamine 2,3-dioxygenase) restores IL-2 production and allows for increased proliferation of CD8 + T cells present in the tumor microenvironment. Anti-PD1 therapy appeared to reject the tumor by allowing antigen-specific vaccine-generated CD8 + T cells to rescue and allowing proliferation. These data suggest that the PD-1 / PD-L1 axis regulates activated tumor-specific T cells.

In clinical trials of melanoma, non-small cell lung cancer, and renal cell carcinoma, anti-tumor response was seen in some patients with anti-PD-1 blocked. Significant advantages due to PD-I inhibition have also been described in the case of advanced melanoma, non-small cell lung cancer, prostate cancer, renal cell cancer, bladder cancer, and colorectal cancer. Studies in the murine model have applied this evidence for glioma treatment. A decrease in tumor-invasive Treg and an increase in survival rate were reported when the combination therapy of IDO, CTLA-4, and PD-L1 inhibitor was administered.

Some PD-1 inhibitors are currently being tested in clinical trials. CT-011 is a humanized IgG1 monoclonal antibody to PD-1. Phase II clinical trials have recently been completed in subjects with diffuse large B-cell lymphoma (DLBCL) undergoing autologous stem cell transplantation. Preliminary results showed that at the end of the follow-up period, 70% of the subjects were unprocessed and 62% of the subjects were alive compared to 62% of the control subjects, compared to 47% of the control subjects. This study revealed that CT-011 not only blocks PD-1 function, but also increases the activity of natural killer cells, thereby enhancing the antitumor immune response.

3의 부작용은 대상체의 14%에서 발생하였다.BMS 936558 is a complete human IgG4 monoclonal antibody that targets the PD-I formulation. In phase I trials, bi-weekly administration of BMS-936558 in subjects with advanced, intractable malignancy showed continued partial or complete degeneration. The most prominent response rates were observed in subjects with melanoma (28%) and renal cell carcinoma (27%), but substantial clinical activity was also observed in subjects with non-small cell lung cancer (NSCLC) . This was also relatively well tolerated; Rating

3 occurred in 14% of the subjects.

BMS 936559 is a complete human IgG4 monoclonal antibody that targets PD-L1, a PD-1 ligand. Phase I results showed that biweekly administration of this drug induced a sustained response, particularly in subjects with melanoma. Objective response rates ranged from 6% to 17%, depending on the cancer type of the subject with progressive-stage NSCLC, melanoma, RCC, or ovarian cancer, and some subjects experienced responses lasting more than one year.

MK 3475 is a 5-part study evaluating the administration, safety and tolerability of drugs in advanced, locally advanced, or metastatic carcinoma, melanoma, or subjects with NSCLC. Humanized IgG4 anti-PD-1 It is a monoclonal antibody.

MPDL 3280A is associated with the presence or absence of chemotherapy in subjects with advanced solid tumors, with bevacizumab targeting the vascular endothelial growth factor receptor (VEGFR), and with BRAF V600-mutant metastatic melanoma in BRAF It is a monoclonal antibody that also targets PD-L1, which has been subjected to Phase I trials with the inhibitor vemurafenib.

AMP 224 is a fusion protein of the extracellular domain of PD-L2, which is the second PD-1 ligand and IgG1, which are likely to block PD-L2 / PD-1 interaction. AMP-224 is currently undergoing Phase I trials as a monotherapy in subjects with currently advanced cancer.

Medi 4736 is an anti-PD-L1 antibody in phase I clinical trials in subjects with advanced malignant melanoma, renal cell carcinoma, NSCLC, and colorectal cancer.

CTLA4 (a cytotoxic T-lymphocyte-related protein) is a protein receptor that down-modulates the immune system. CTLA4 is found on the surface of T cells and causes a cellular immune attack on the antigen. T cell attack can be triggered by stimulating CD28 receptors on T cells. T cell attack can be disrupted by stimulating the CTLA4 receptor. Ipilimumab (YERVOY®), an innovative new drug immunotherapy that is a monoclonal antibody that targets CTLA-4 on the surface of T cells, was intended for the treatment of melanoma.

Preferably, the invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutic agent in combination with a drug-resistant immunocompetent cell and an immune checkpoint inhibitor. Preferably, the checkpoint inhibitor is a biological therapeutic agent or a small molecule. Preferably, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a complete human antibody, a fusion protein, or a combination thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, OX40, A2aR, B-7 family ligands, or combinations thereof.

Preferably, the therapeutic agent is a chemotherapeutic agent, an immunostimulant, a T cell growth factor, an interleukin, an antibody, a vaccine or any combination thereof. Preferably, the interleukin is IL-7 or IL-15. Preferably, the interleukin is glycated IL-7.

Preferably, the drug-tolerant immunocompetent cell, therapeutic agent, and checkpoint inhibitor are administered simultaneously or sequentially, either sequentially, one after the other, or the other two simultaneously, or in any order subsequent to the second followed by the third . Preferably, the drug-resistant immunocompetent cell is administered prior to the therapeutic agent and the immune checkpoint inhibitor. Preferably, the checkpoint inhibitor is a PD-I inhibitor or a CTLA-4 inhibitor.

Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises gamma delta T-cells, NK cells, or any combination thereof. Preferably, the population of cytotoxic immune cells is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% Of γδ T-cells. Preferably, the population of cytotoxic immune cells comprises about 50% to about 95% of the gamma delta T-cells. Preferably, the population of cytotoxic immune cells used in the compositions and methods of the invention comprises gamma delta T-cells and natural killer (NK) cells. Preferably, the population of cytotoxic immune cells comprises about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% . Preferably, the population of cytotoxic immune cells comprises about 5% to about 25% of NK cells.

Preferably, the isolated compositions of cytotoxic immune cells used in the compositions and methods of the invention comprise gamma delta T-cells, NK cells or any combination thereof and optionally monocytes, macrophages and dendritic cells, , Greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50% or more of cytotoxic immune cells present in the composition The excess expresses a polypeptide conferring resistance to the chemotherapeutic agent. Preferably, the isolated composition of cytotoxic immune cells used in accordance with the present invention comprises gamma delta T-cells, NK cells or any combination thereof, wherein at least about 50% of the gamma delta T-cells (when present) More than about 50% of NK cells (when present) express polypeptides that confer resistance to chemotherapeutic agents. Preferably, a polypeptide that imparts resistance to the chemotherapeutic agents is O ⁶ methyl guanine DNA methyl transferase (MGMT), dihydro folate drug-resistant variants of the reductase (L22Y-DHFR), thymidine delay tree synthetase, multidrug Resistant < / RTI > protein (MDR1).

Preferably, the invention provides monocytes, macrophages and dendritic cells that are genetically engineered to express a polypeptide comprising a chemotherapeutic agent and a gamma delta T-cell, an NK cell, or any combination thereof and additionally a resistance to a chemotherapeutic agent Comprising administering to a subject in need thereof an immunologic checkpoint inhibitor together with a chemotherapeutic resistant composition of an immunocompetent cell, optionally including other immunocompetent cells, including, but not limited to, < RTI ID = 0.0 > do. In vitro production and proliferation of a composition comprising a drug-resistant gamma delta T-cell, an NK cell or any combination thereof, enables the administration of an immunocompetent cell-based therapy concurrently with chemotherapy and checkpoint inhibitors in such an environment Can potentially improve tumor clearance while establishing and maintaining anti-tumor immunity, which may lead to prolonged tumor removal for a long time. Preferably, the immunocompetent is genetically engineered to express at least one, at least two, at least three or more different tumor-bearing moieties, each tumor-bearing moiety recognizing a tumor antigen.

Preferably, the invention provides a method of treating cancer, and in particular cancerous tumors. The methods of the present invention combine the use of immunotherapy and immune checkpoint inhibitors with chemotherapeutic agents capable of killing cancerous cells or reducing their proliferation in order to effectively remove cancerous cells that cause drug resistance or otherwise avoid chemotherapeutic agents . The method of the invention may be used to treat cells comprising, but not limited to, gamma delta T-cells from the treated patient or other source, for example, as described by Lamb LS et al. In U.S. Patent 7,078,034, Lt; RTI ID = 0.0 > immune < / RTI > cells. The isolated cell may then be transfected with a nucleic acid vector comprising a heterologous nucleotide sequence encoding a polypeptide that confers resistance to the selected chemotherapeutic agent. Thereafter, the cancer, and particularly a patient in need of tumor therapy, can be administered a dose or dose of the T-cell transfected before, after, or together with the chemotherapeutic agent and the checkpoint inhibitor. The agent itself, which is toxic to the targeted cancer cells and which reduces cell proliferation and viability, also induces the microenvironment of the tumor to reduce local tumor-induced immunosuppression and reduce cell migration into the tumor, as well as cell surface Lt; RTI ID = 0.0 > stress-related < / RTI > For example, transfected < RTI ID = 0.0 > T-cells < / RTI >

Preferably, genetically modified cytotoxic immune cells, including gamma delta T-cells, NK cells, or a combination thereof, are used to treat, for example, but not limited to, direct injection into a tumor mass, delivery to a tumor entering the tumor mass , ≪ / RTI > or a combination of both. For example, it is contemplated that cells may be delivered directly to a gluoblastoma mass in the patient's brain by direct grafting through a tumor mass, intracavity after ablation, cannula needle inserted intra-abdominally or deeply, or catheter .

Preferably, the cytokine (IL-2, IL-12, GM-CSF, etc.) is delivered to the patient in some protocols that combine immunotherapy and chemotherapy to treat cancer in a subject human or animal patient, The formation of lymphocytes can be boosted. In another method, an anti-CD137 specific antibody can be used. CD137 is a member of the tumor necrosis factor (TNF) / nerve growth factor (NGF) receptor family and is expressed by activated T- and B-lymphocytes and monocytes; Its ligand has been found to play an important role in the regulation of the immune response. The anti-CD137 monoclonal antibody specifically binds to CD137-expressing immune cells, such as activated T-cells and newly isolated mouse dendritic cells (DCs), resulting in an immune response to tumor cells, particularly cytotoxic T cell responses Lt; / RTI >

Preferably, the present invention provides a method of obtaining a population of cytotoxic immune cells, wherein said isolated cytotoxic immune cells are genetically modified to be resistant to the therapeutic; Administering an effective amount of a therapeutic agent to a patient in need thereof; Administering to the patient a population of isolated genetically modified cytotoxic immune cells, such that the cytotoxic immune cells are transferred to the tumor; And an effective amount of an immune checkpoint inhibitor to the patient to reduce the cancer in the patient.

Preferably, the cytotoxic immune cell is a γδ T-cell. Preferably, the cytotoxic immune cells are gamma delta T-cells and natural killer (NK) cells. Preferably, the cytotoxic immune cells are gamma delta T-cells, NK cells and may further comprise other immunologically competent cells, including but not limited to monocytes, macrophages and dendritic cells. Preferably, the cytotoxic immune cells can be isolated from patients with cancer. Preferably, the cytotoxic immune cells can be isolated from a source other than a cancer patient.

Preferably, the cytotoxic immune cells are gamma delta T-cells derived from human induced pluripotent stem cells (hiPSC). Preferably, the cytotoxic immune cells are gamma delta T-cells and NK cells derived from human induced pluripotent stem cells (hiPSC). Preferably, the cytotoxic immune cells are derived from human induced pluripotent stem cells (hiPSC) and are further gamma delta T-cells, NK cells and other immunocompetent cells, including but not limited to monocytes, macrophages and dendritic cells . Preferably, pluripotent stem cells can be isolated from patients with cancer. Preferably, pluripotent stem cells can be isolated from sources other than cancer patients.

Preferably, the therapeutic agent is characterized by cells that exhibit resistance to the therapeutic agent when the cell receives the heterologous nucleic acid and when the heterologous nucleic acid is expressed in the cell. Preferably, the therapeutic agent can be a cytotoxic chemotherapeutic agent selected from the group consisting of: alkylating agents (e.g., cyclophosphamide, ifosfamide); Metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or derivatives thereof); DNA demethylating agents (also known as antimetabolites; for example, azacytidine); Substituted nucleotides; Substituted nucleosides; Antitumor antibiotics (e. G., Mitomycin, adriamycin); Plant-derived antineoplastic agents (e. G., Vincristine, vindosine, TAXOL, paclitaxel, ablactic acid); Cisplatin; Carboplatin; Etoposide, and the like. Such agents include the anticancer agents trimethotrexate (TMTX); Temozolomide; Ralitriptycide; S- (4-nitrobenzyl) -6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); Nitrosoureas [e.g., bis-chloronitrosourea (BCNU) (Carmustine), rosmutin (CCNU) +/- procarbazine and vincristine (PCV therapy), potemustine]; Cytarabine; And camptothecin; ≪ / RTI > and any therapeutic derivatives thereof.

Preferably, the step of obtaining a population of cytotoxic immune cells that are genetically modified to be resistant to the therapeutic agent is performed by, for example, obtaining a biological sample that includes, without limitation, a tissue sample comprising blood or a tumor biopsy from the subject To obtain a population of cytotoxic immune cells from a subject, such as a human or animal subject. The sample may be selectively enriched for cytotoxic immune cells and other immunocompetent cells and / or cells present in the sample may be selectively propagated to increase the population of cells present in the sample. The cell is preferably stably transfected or genetically edited with a vector comprising a heterologous nucleic acid sequence operably linked to a promoter, wherein said heterologous nucleic acid sequence imparts cell resistance to one or more therapeutic agents, such as a chemotherapeutic agent Lt; / RTI > Preferably, the population of stably transfected cytotoxic immune cells can remain viable.

Preferably, the therapeutic agent may be trimethotrexate or methotrexate, and the heterologous nucleic acid sequence encodes a dihydrofolate reductase or derivative thereof. Preferably, the therapeutic agent may be temozolomide or a therapeutically active derivative thereof, and the heterologous nucleic acid sequence may encode an O ⁶ methylguanine DNA methyltransferase or derivative thereof. Preferably, the chemotherapeutic agent may be cisplatin, doxorubicin or paclitaxel or a therapeutically active derivative thereof, and the heterologous nucleic acid sequence may encode a multidrug resistance-1 protein (MDR1) or a derivative thereof.

Preferably, the isolated genetically modified cytotoxic immune cells, therapeutic agents, and immuno checkpoint inhibitors can be co-administered to the patient. Preferably, genetically modified cytotoxic immune cells, therapeutic agents, and immuno checkpoint inhibitors can be administered sequentially to the patient. Preferably, genetically modified cytotoxic immune cells, therapeutic agents, and immuno checkpoint inhibitors can be administered simultaneously to the patient. Preferably, genetically modified cytotoxic immune cells are administered to the patient directly into the tumor or into the proximal vein and into the tumor. Preferably, the tumor is a glioblastoma.

Preferably, the immune checkpoint inhibitor may be a biological therapeutic agent or a small molecule. Preferably, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a complete human antibody, a fusion protein, an antigen-binding fragment, or a combination thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or a combination thereof. Exemplary immuno checkpoint inhibitors include, but are not limited to, tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK- PBL monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody) Antibody), MSB0010718C (anti-PD1 antibody) and YERVOY® / eicilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

Preferably, the invention provides the use of an immuno checkpoint inhibitor drug class that inhibits CTLA-4. Suitable anti-CTLA4 antagonists for use in the methods of the present invention include anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, CTLA4 antibody, a polyclonal anti-CTLA4 antibody, a chimeric anti-CTLA4 antibody, MDX-010 (eicilimumab), tremelimumum, anti-CD28 antibody, anti-CTLA4 adenectin, -CTLA4 fragment, a heavy chain anti-CTLA4 fragment, a light chain anti-CTLA4 fragment, and an inhibitor of CTLA4 that acts on a co-stimulatory pathway. Preferably, the additional anti-CTLA4 antagonists include, but are not limited to, the ability of the CD28 antigen to destroy its ability to bind its cognate ligand, inhibit the ability of CTLA4 to bind its cognate ligand, To increase the T cell response through the stimulatory pathway, to destroy the ability of B7 to bind to CD28 and / or CTLA4, to destroy the ability to activate the co-stimulatory pathway of B7, and to bind CD28 CD28 and / or CTLA4 Destroys the ability to destroy the ability to activate the co-stimulatory pathway of CD86, destroy the ability to bind to CD28 and / or CTLA4, destroy the ability to activate the co-stimulatory pathway of CD86, - Any inhibitor capable of destroying the stimulation path normally activated. It is essential that among other anti-CTLA4 antagonists, among other members of the co-stimulatory pathway are small molecule inhibitors of CD28, CD80, CD86, CTLA4; Among other members of the co-stimulatory pathway are antibodies directed against CD28, CD80, CD86, CTLA4; Among other members of the co-stimulatory pathway are antisense molecules directed against CD28, CD80, CD86, CTLA4; Of the other members of the co-stimulatory pathway are the RNAi inhibitors of CD28, CD80, CD86, CTLA4 (both single and double stranded), among the other members of the adnexin, co-stimulatory pathway directed against CD28, CD80, CD86, CTLA4, All).

Preferably, immunostimulants, T cell growth factors and interleukins can be used in combination with checkpoint inhibitors and drug-resistant immunotherapy. Immune stimulants are substances (drugs and nutrients) that stimulate the immune system by inducing activation of any of its components or by increasing activity. Immunostimulants include, but are not limited to, bacterial vaccines, colony stimulating factors, interferons, interleukins, other immunostimulants, therapeutic vaccines, vaccine combinations and viral vaccines. T cell growth factor is a protein that stimulates the proliferation of T cells. Examples of T cell growth factors include IL-2, IL-7, IL-15, IL-17, IL21 and IL-33.

Preferably, the present invention provides a composition comprising at least one checkpoint inhibitor, and a separate population of cytotoxic immune cells comprising a gamma delta T-cell, an NK cell, or any combination thereof, wherein the cell More than about 5% and preferably greater than about 50% of the population of toxic immune cells express polypeptides that confer resistance to chemotherapeutic agents. Preferably, the composition provides a composition comprising at least one checkpoint inhibitor, and a separate population of cytotoxic immune cells comprising gamma delta T-cells and NK cells, wherein the concentration of the drug in a population of cytotoxic immune cells About 50% to about 95% are gamma delta T-cells and about 5% to about 25% of the population of cytotoxic immune cells are NK cells.

Preferably, the therapeutic agent, immuno checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition as disclosed herein is administered orally or parenterally, for example, intravenously, intramuscularly, subcutaneously, orally, intraocularly, By passive or accelerated absorption through the skin using various routes including intraperitoneal, rectal, aquatic, intratumoral, intraduodenal, intradermal or by using, for example, skin patches or percutaneous iontophoresis, respectively Can be administered to an individual. The therapeutic agent, checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition may also be administered intravenously or intraarterially into the site of the pathological condition, for example, into a blood vessel that is supplied to the tumor.

Preferably, the immune checkpoint inhibitor is administered in an amount of less than 0.0001 mg / kg, 0.0001-0.001 mg / kg, 0.001-0.01 mg / kg, 0.01-0.05 mg / kg, 0.05-0.1 mg / kg, 0.1-0.2 mg / 0.3-0.5 mg / kg, 0.5-0.7 mg / kg, 0.7-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, 5 mg / kg, 5-6 mg / kg, 7-8 mg / kg, 8-9 mg / kg, 9-10 mg / kg, at least 10 mg / ≪ / RTI > Preferably, the checkpoint inhibitor is administered at least once a week, at least twice a week, at least three times a week, at least once every two weeks, or at least once every month or every several months. Preferably, the checkpoint inhibitor is administered in a single dose, two doses, three doses, four doses, five doses, or six doses or more.

Preferably, the present invention encompasses a cytotoxic therapeutic agent having the property of suppressing the survival of cancer cells, a separate population of cytotoxic immune cells genetically modified so that the cytotoxic immune cells are resistant to the therapeutic agent, and an immunocompromised checkpoint inhibitor The present invention provides a system for treating cancer in a patient suffering from cancer. Preferably, the population of cytotoxic immune cells comprises gamma delta T-cells, NK cells or any combination thereof. Preferably, the population of cytotoxic immune cells comprises gamma delta T-cells and natural killer cells and optionally comprises other immunocompetent cells. Preferably, the population of cytotoxic immune cells may comprise a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic acid sequence encodes a polypeptide that confers resistance to the therapeutic agent upon expression in the cell . Preferably, the therapeutic agent is a cytotoxic chemotherapeutic agent selected from the group consisting of: alkylating agents (e.g., cyclophosphamide, ifosfamide); Metabolic antagonists (e.g., methotrexate (MTX), 5-fluorouracil or derivatives thereof); DNA demethylating agents (also known as antimetabolites; for example, azacytidine); Substituted nucleotides; Substituted nucleosides; Antitumor antibiotics (e. G., Mitomycin, adriamycin); Plant-derived antineoplastic agents (e. G., Vincristine, vindosine, TAXOL, paclitaxel, ablactic acid); Cisplatin; Carboplatin; Etoposide, and the like. Such agents include the anticancer agents trimethotrexate (TMTX); Temozolomide; Ralitriptycide; S- (4-nitrobenzyl) -6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); Nitrosoureas [e.g., bis-chloronitrosourea (BCNU) (Carmustine), rosmutin (CCNU) +/- procarbazine and vincristine (PCV therapy), potemustine]; Cytarabine; And camptothecin; ≪ / RTI > and any therapeutic derivatives thereof. Preferably, the therapeutic agent is trimethotrexate or methotrexate, and the heterologous nucleic acid sequence encodes a dihydrofolate reductase or derivative thereof. Preferably, the therapeutic agent is temozolomide or a therapeutic agent derivative thereof, and the heterologous nucleic acid sequence encodes O ⁶ methylguanine DNA methyltransferase (MGMT) or a derivative thereof. Preferably, the therapeutic agent is nitroso urea or a therapeutic agent derivative thereof, and the heterologous nucleic acid sequence encodes O ⁶ methyl guanine DNA methyltransferase (MGMT) or a derivative thereof.

Preferably, the immune checkpoint inhibitor is a biological therapeutic agent or a small molecule. Preferably, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a complete human antibody, a fusion protein, an antigen-binding fragment, or a combination thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or a combination thereof. Exemplary immuno checkpoint inhibitors include, but are not limited to, tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK- PBL monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody) Antibody), MSB0010718C (anti-PD1 antibody) and YERVOY® / eicilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

Preferably, the invention includes the use of a checkpoint inhibitor drug class that inhibits CTLA-4. Suitable anti-CTLA4 antagonists for use in the methods of the present invention include anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, CTLA4 antibody, a polyclonal anti-CTLA4 antibody, a chimeric anti-CTLA4 antibody, MDX-010 (eicilimumab), tremelimumum, anti-CD28 antibody, anti-CTLA4 adenectin, -CTLA4 fragment, a heavy chain anti-CTLA4 fragment, a light chain anti-CTLA4 fragment, and an inhibitor of CTLA4 that acts on a co-stimulatory pathway.

Preferably, the additional anti-CTLA4 antagonists include, but are not limited to, the ability of the CD28 antigen to destroy its ability to bind its cognate ligand, inhibit the ability of CTLA4 to bind its cognate ligand, To increase the T cell response through the stimulatory pathway, to destroy the ability of B7 to bind to CD28 and / or CTLA4, to destroy the ability to activate the co-stimulatory pathway of B7, and to bind CD28 CD28 and / or CTLA4 Destroys the ability to destroy the ability to activate the co-stimulatory pathway of CD86, destroy the ability to bind to CD28 and / or CTLA4, destroy the ability to activate the co-stimulatory pathway of CD86, - Any inhibitor capable of destroying the stimulation path normally activated. It is essential that among other anti-CTLA4 antagonists, among other members of the co-stimulatory pathway are small molecule inhibitors of CD28, CD80, CD86, CTLA4; Among other members of the co-stimulatory pathway are antibodies directed against CD28, CD80, CD86, CTLA4; Among other members of the co-stimulatory pathway are antisense molecules directed against CD28, CD80, CD86, CTLA4; Of the other members of the co-stimulatory pathway are the RNAi inhibitors of CD28, CD80, CD86, CTLA4 (both single and double stranded), among the other members of the adnexin, co-stimulatory pathway directed against CD28, CD80, CD86, CTLA4, All).

Preferably, immunostimulants, T cell growth factors and interleukins can be used. Immune stimulants are substances (drugs and nutrients) that stimulate the immune system by inducing activation of any of its components or by increasing activity. Immunostimulants include, but are not limited to, bacterial vaccines, colony stimulating factors, interferons, interleukins, other immunostimulants, therapeutic vaccines, vaccine combinations and viral vaccines. T cell growth factor is a protein that stimulates the proliferation of T cells. Examples of T cell growth factors include IL-2, IL-7, IL-15, IL-17, IL21 and IL-33.

Preferably, the therapeutic agent, checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition as disclosed herein is administered orally or parenterally, for example, intravenously, intramuscularly, subcutaneously, orally, intraocularly, intraocularly, intraperitoneally By passive or accelerated absorption through the skin using various routes including, for example, intravenous, intramuscular, intratracheal, intratracheal, intraduodenal, intradermal, or using, for example, skin patches or percutaneous iontophoresis, Lt; / RTI > The therapeutic agent, checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition may also be administered intravenously or intraarterially into the site of the pathological condition, for example, into a blood vessel that is supplied to the tumor.

Preferably, the total amount of agent administered in the practice of the methods of the invention may be administered to the subject as a single dose, either as a bolus or by a relatively short period of time, or may be administered using a fractionated therapy protocol And multiple doses are administered over a prolonged period of time. Those skilled in the art will appreciate that the amount of the composition for treating a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan will adjust the specific capacity as needed.

Preferably, the checkpoint inhibitor is present in an amount of less than 0.0001 mg / kg, 0.0001-0.001 mg / kg, 0.001-0.01 mg / kg, 0.01-0.05 mg / kg, 0.05-0.1 mg / 0.3 mg / kg, 0.3-0.5 mg / kg, 0.5-0.7 mg / kg, 0.7-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / 5 mg / kg, 5-6 mg / kg, 7-7 mg / kg, 7-9 mg / kg, 9-10 mg / kg, at least 10 mg / Administered in any combination. Preferably, the checkpoint inhibitor is administered at least once a week, at least twice a week, at least three times a week, at least once every two weeks, or at least once every month or every several months. Preferably, the checkpoint inhibitor is administered in a single dose, two doses, three doses, four doses, five doses, or six doses or more.

Preferably, the present invention relates to a therapeutic agent having a property of inhibiting the survival of cancer cells and inducing a stress protein in cancer cells, a cytotoxic immune cell comprising γδ T-cells, NK cells, or any combination thereof, A system for treating glioblastoma in a patient comprising a separate population of cytotoxic immune cells genetically modified so that a population of cytotoxic immune cells are resistant to the therapeutic agent, optionally comprising other immunocompetent cells, and a checkpoint inhibitor, to provide. Preferably, the population of cytotoxic immune cells comprises a heterologous nucleic acid sequence operably linked to a promoter, wherein the heterologous nucleic acid sequence encodes a polypeptide that confers resistance to the therapeutic agent upon expression in the cell.

Preferably, the therapeutic agent is a cytotoxic chemotherapeutic agent selected from the group consisting of an alkylating agent, a metabolic antagonist, an antitumor antibiotic, and a plant-derived antitumor agent.

Preferably, the therapeutic agent is selected from the group consisting of trimethotrexate (TMTX), methotrexate (MTX), temozolomide, ralitriptycide, S- (4-nitrobenzyl) -6-thioinosine (NBMPR), camptothecin, 6-benzylguanidine , Cytarabine, and any therapeutic derivatives thereof.

Preferably, the therapeutic agent is trimethotrexate or methotrexate, and the heterologous nucleic acid sequence encodes a dihydrofolate reductase or derivative thereof.

Preferably, the immune checkpoint inhibitor may be a biological therapeutic agent or a small molecule. Preferably, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a complete human antibody, a fusion protein, an antigen-binding fragment, or a combination thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof. Preferably, the checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- , CHK2, A2aR, OX40, B-7 family ligands, or a combination thereof. Exemplary immuno checkpoint inhibitors include, but are not limited to, tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK- PBL monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody) Antibody), MSB0010718C (anti-PD1 antibody) and YERVOY® / eicilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include but are not limited to PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

Preferably, the invention encompasses the use of a specific class of immuno checkpoint inhibitor drugs that inhibit CTLA-4. Suitable anti-CTLA4 antagonists for use in the methods of the present invention include anti-CTLA4 antibodies, human anti-CTLA4 antibodies, mouse anti-CTLA4 antibodies, mammalian anti-CTLA4 antibodies, humanized anti-CTLA4 antibodies, CTLA4 antibody, a polyclonal anti-CTLA4 antibody, a chimeric anti-CTLA4 antibody, MDX-010 (eicilimumab), tremelimumum, anti-CD28 antibody, anti-CTLA4 adenectin, -CTLA4 fragment, a heavy chain anti-CTLA4 fragment, a light chain anti-CTLA4 fragment, and an inhibitor of CTLA4 that acts on a co-stimulatory pathway.

Preferably, the additional anti-CTLA4 antagonists include, but are not limited to, the ability of the CD28 antigen to destroy its ability to bind its cognate ligand, inhibit the ability of CTLA4 to bind its cognate ligand, To increase the T cell response through the stimulatory pathway, to destroy the ability of B7 to bind to CD28 and / or CTLA4, to destroy the ability to activate the co-stimulatory pathway of B7, and to bind CD28 CD28 and / or CTLA4 Destroys the ability to destroy the ability to activate the co-stimulatory pathway of CD86, destroy the ability to bind to CD28 and / or CTLA4, destroy the ability to activate the co-stimulatory pathway of CD86, - Any inhibitor capable of destroying the stimulation path normally activated. It is essential that among other anti-CTLA4 antagonists, among other members of the co-stimulatory pathway are small molecule inhibitors of CD28, CD80, CD86, CTLA4; Among other members of the co-stimulatory pathway are antibodies directed against CD28, CD80, CD86, CTLA4; Among other members of the co-stimulatory pathway are antisense molecules directed against CD28, CD80, CD86, CTLA4; Of the other members of the co-stimulatory pathway are the RNAi inhibitors of CD28, CD80, CD86, CTLA4 (both single and double stranded), among the other members of the adnexin, co-stimulatory pathway directed against CD28, CD80, CD86, CTLA4, All).

Preferably, immunostimulants, T cell growth factors and interleukins can be used. Immune stimulants are substances (drugs and nutrients) that stimulate the immune system by inducing activation of any of its components or by increasing activity. Immunostimulants include, but are not limited to, bacterial vaccines, colony stimulating factors, interferons, interleukins, other immunostimulants, therapeutic vaccines, vaccine combinations and viral vaccines. T cell growth factor is a protein that stimulates the proliferation of T cells. Examples of T cell growth factors include IL-2, IL-7, IL-15, IL-17, IL21 and IL-33.

Preferably, the therapeutic agent, immuno checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition as disclosed herein is administered orally or parenterally, for example, intravenously, intramuscularly, subcutaneously, orally, intraocularly, By passive or accelerated absorption through the skin using various routes including intraperitoneal, rectal, aquatic, intratumoral, intraduodenal, intradermal or by using, for example, skin patches or percutaneous iontophoresis, respectively Can be administered to an individual. The therapeutic agent, checkpoint inhibitor, biological therapeutic agent or pharmaceutical composition may also be administered intravenously or intraarterially into the site of the pathological condition, for example, into a blood vessel that is supplied to the tumor.

Preferably, the total amount of the agent to be administered in the practice of the methods of the present invention may be administered to the subject as a single dose, as a bolus or over a relatively short period of time, or administered using a fractionated therapy protocol And the multiple doses are administered over a prolonged period of time. Those skilled in the art will appreciate that the amount of the composition for treating a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan will adjust the specific capacity as needed.

Preferably, the checkpoint inhibitor is present in an amount of less than 0.0001 mg / kg, 0.0001-0.001 mg / kg, 0.001-0.01 mg / kg, 0.01-0.05 mg / kg, 0.05-0.1 mg / 0.3 mg / kg, 0.3-0.5 mg / kg, 0.5-0.7 mg / kg, 0.7-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / 5 mg / kg, 5-6 mg / kg, 7-7 mg / kg, 7-9 mg / kg, 9-10 mg / kg, at least 10 mg / Administered in any combination. Preferably, the checkpoint inhibitor is administered at least once a week, at least twice a week, at least three times a week, at least once every two weeks, or at least once every month or every several months. Preferably, the checkpoint inhibitor is administered in a single dose, two doses, three doses, four doses, five doses, or six doses or more.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, so that not only the numerical values explicitly recited as ranges of limits, but also each individual value contained within that range, as each numerical value and sub- Value, or sub-range of a given value. For illustrative purposes, a concentration range of "about 0.1% to about 5%" is intended to include not only explicitly stated concentrations of from about 0.1 wt% to about 5 wt%, but also individual concentrations within the indicated ranges (e.g., 2%, 3%, and 4%) and sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%). The term "about" means that the numerical value (s) to be modified is at least one of: ± 1%, ± 2%, ± 3%, ± 4%, ± 5%, ± 6%, ± 7%, ± 8% ± 10% or more. Also, the phrases "about " x 'to " y" include from about ' x to about ' y ' '.

Many variations and modifications may be made to the invention described above without departing substantially from the spirit and principles of the invention. All such variations and modifications are intended to be included within the scope of the present invention and protected by the following claims.

Example

The following examples are provided for illustration and should not be construed as limiting the invention in any way.

Example 1 - Cancer Therapy Paradigm

A cancer patient is treated using a combination therapy comprising a chemotherapeutic agent, a drug-resistant γδ-T cell and / or a drug-resistant natural killer cell, and an immuno checkpoint inhibitor (s). The chemotherapeutic agent may be selected from the group consisting of an alkylating agent (e.g., cyclophosphamide, iosp sporid), a metabolic antagonist (e.g., methotrexate (MTX), 5- fluorouracil or a derivative thereof), an antitumor antibiotic Selected from plant-derived antineoplastic agents (e. G., Vincristine, vindocin, TAXOL, paclitaxel, ablactic acid), cisplatin, carboplatin, etoposide, and the like. Such formulations include, but are not limited to, the anticancer agents trimethotrexate (TMTX), temozolomide, ralitriptycide, S- (4-nitrobenzyl) -6-thioinosine (NBMPR), 6-benzylguanidine Urea (BCNU), cytarabine, and camptothecin, or any therapeutic derivatives thereof. Drug-resistant γδ-T cells and natural killer cells are resistant to chemotherapeutic agents. (B7-H1, CD274), PDL2 (B7-DC, CD273), PD1, B7- H3 (CD276), B7-H4 (B7-S1, B7x, VCTN1), BTLA (CD272), HVEM, TIM3 (HAVcr2), GAL9, LAG3 (CD223), VISTA, KIR, 2B4 CD15 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinase, OX40, A2aR and various B-7 family ligands, which are expressed on all NK, gamma delta and memory CD8 ⁺ Inhibitors.

Evaluation of immunotherapeutic activity with chemotherapy

The response criteria include an evaluation based on response criteria for solid tumors (World Health Organization Offset Publication No. 48, 1979); RECIST (Response Evaluation Criteria in Solid Tumors; J Natl Cancer Inst 2000; 92: 205-16; Eisenhauer EA et al., Eur J Cancer 2009; 45: 228-47); Immune-Related Response Criteria (Jedd WE et al., Clin Cancer Res 2009; 15 (23), 7412-7420); and iRANO (Immunotherapy Response Assessment in Neuro-Oncology; Okada H et al., Lancet Oncol 2015 Nov; 16 (15): e534-42).

result

Chemotherapy as a combination therapy further comprising a drug-resistant γδ-T cell (and / or drug-resistant natural killer cell) and an inhibitor (s) of an immunocompromised protein may be used for an improved or extended anti-tumor response; Medically beneficial response; Additional or synergistic anti-tumor activity, as compared to chemotherapy alone or in combination with chemotherapy in combination with drug-resistant < RTI ID = 0.0 > [gamma] < / RTI > -δ T cells (and / or drug-resistant natural killer cells); Contraction of baseline lesions without new lesions; Persistent stable disease in which the total tumor burden may steadily decrease; Increased post - tumor burden; And reactions in the presence of new lesions. Other clinical outcomes include an increase in tumor invasive lymphocytes as measured by an increase in tumor size; And promoting long-term survival.

For example, temozolomide (TMZ) -treated tumors express an increased number of stress ligands on the surface of tumor cells, such as MICA, MICB, and ULBP1-6. Tumor cell death is promoted by drug-resistant γδ-T cells and / or drug-resistant natural killer cells, and its receptor (s), eg, NKG2D, recognize tumor-specific stress ligands. However, tumor cells can enzymatically cleave MICA and MICB to release soluble MICA and MICB. When bound by an effector cell receptor in plasma, this can lead to downregulation of receptor expression leading to immunodeficiency. Chemotherapeutic agents such as TMZ have been shown to increase the cell surface expression of stress ligands on tumor tissues without significantly increasing the concentration of soluble ligands in plasma. Reactants to antibody inhibitors of CTLA-4 showed the ability to cause a humoral response to NKG2D ligands such as MICA. Such a response reduces soluble MICA, which allows the expression of amplified stress ligand on the tumor surface, thereby enhancing tumor cell death by drug-resistant < RTI ID = 0.0 > [gamma] < / RTI >

Example 2 - Analysis of checkpoint molecule expression and function in glioma and γδ T cells

Checkpoint molecule expression was assessed on GMP-treated γδ T cells and glioblastoma tumor cells, and the function of concentrated γδ T cells was assessed in vitro.

Way

In vitro expansion of γδ T cells

Human collections from healthy donors A collection of products was purchased from Hemacare (Van Nuys CA). The product was moved to the UAB Cell Therapy Laboratory GMP facility. For static cultivation: All operations were performed in a Class 100 laminar flow hood surrounded by Class 10K / ISO 7 Classified Airflows. The product was diluted with HBSS (Hank's balanced salt solution) (v / v) and mononuclear cells were separated by density gradient separation on Ficoll (Sigma-Aldrich; St. Louis, Mo.). Liver was harvested and washed twice in HBSS. The cell pellet was resuspended in CTS ™ OpPTmizer MIZER ™ T cell proliferation serum free medium (ThermoFisher Scientific; Waltham, MA) supplemented with 2 mM zoledronic acid (Novartis; Basel HV) and 100 u / mL IL2 (Miltenyi Biotec Ltd; Bergisch Gladbach, Germany) It was resuscitated. Cells were cultured in standard T150 or T75 flasks. For closed system incubation: All work, including preparation of media, stocks and buffers, was performed using GMP grade reagents under the bio-safety workbench in an ISO 7 clean room. Automated Ficoll isolation and culture of the product was performed in a CLINIMACS PRODIGY® (Miltenyi Biotec Ltd; Bergisch Gladbach, Germany) bioreactor using a custom cell treatment program and a TS520 tubing set. Cells were then plated on a CentriCult-Unit of CLINIMACS PRODIGY® using a combination (ZOL / IL-2) of Zometa (Novartis, Basel) and Interleukin-2 (Miltenyi Biotec Ltd; Bergisch Gladbach, Germany) And grown in OpTmizer serum-free culture medium (Thermo Fisher Scientific; Waltham, Mass.). Cultures were maintained at a density of 1-2 x 106 cells / ml and supplemented with IL-2 100 u / ml every other day until cells were harvested at day 13. The process was continuously monitored at regular intervals to determine proliferation and phenotypic kinetics as compared to small-scale cultures of flasks. After 13 days of culture, the phenotype of the proliferated γδ T cells was determined and cytotoxicity was determined on standard leukemia cell line and glioma tumor cells.

Flow cytometry

Multi-parameter flow cytometry was used to determine phenotype & functional profiles in donor PBMC and cultured products. Expression of checkpoint molecules and γδ T cell concentrates was monitored using flow cytometry using DuraClone T cell subsets (Beckman Coulter) + anti-PD-1, anti-CTLA-4 and anti-PD-L1 panels. Samples of the product were analyzed by culturing for 1 day and 13 days. The newly isolated PDX-derived glioblastoma tumor cells were analyzed for PD-L1 expression using flow cytometry. Single cell suspensions of PDX-derived glioblastoma cells were blocked with FcR Block (Biolegend) for 15 minutes and then stained with PD-L1 antibody and individual islets (IgG1). The stained cells were obtained on a BD fortessa X-20 flow cytometer.

Evaluation of flow cytometry based on flow cytometry

The efficacy of the cell product was determined using an in vitro cytotoxicity assay for K562 cells. Proliferated γδ T cells were evaluated for cytotoxicity against human leukemia cell line (K562) and tumor cells (JX22T, JX12T and JX59T) at day 13 using flow cytometry-based cytotoxicity assays. The Basic Cytotoxicity Assay Kit (https: // immunochemistry.com) contains two fluorescent reagents, CFSE and 7-AAD. CFSE, a green fluorescent membrane, was used to label target cells (K562). Unfiltered effector cells were added at the target rate with increasing effector, incubated for 4 hours with target cells, and then obtained by adding 7-AAD, a red fluorescent live / dead stain, and using flow cytometry Respectively. Cytotoxicity is calculated as the percentage of green target cells that are the detected red channels. % Cytotoxicity formula = # dead cells / # live cells + # dead cells * 100.

Results & Discussion

PBMC newly isolated from two healthy donors (Donor 1: 043692; Donor 2: 043988) were used for the proliferation of γδ T cells in vitro. Upon investigation of checkpoint expression by flow cytometry, the present inventors noted the upregulation of PD-1 and CTLA-4 and PD-L1 in the proliferated γδ T cells. On day 13, PD-1 increased more than 20% and CTLA-4 expression ranged from 3 to 6%. Interestingly, PD-L1 was also upregulated in both donors (> 9%), [Fig. 1-4]. No significant change in the level of expression of checkpoint molecules was observed in γδ T cells transfected with MGMT compared to untransfected γδ T cells. Moderate downregulation of PD-1 and CTLA-4 in dormant (no IL2 and no ZOL) γδ T cells was also observed compared to stimulated cultures (Fig. 5).

PD-L1 expression in PDX-derived glioma cells was also analyzed by flow cytometry. PD-L1 receptor expression was low or undetectable compared to the A small control. The percentage of PD-L1 expression varied in JX12T (0%), JX22T (1.8%) and JX59T (10.4%) (Fig.

Next, the present inventors investigated how blocking of the immunosuppressive receptor had an effect on the gamma delta T cell ability to increase cytotoxicity against cancer cells (Fig. 7). Gamma T lymphocytes were cultured in a black mixture containing glioma glioma tumor cells (JX22T, JX12T, JX59T) and K562 (a cell line established from chronic myelogenous leukemia) at a concentration of 20 μg / 1 ratio. High levels of cytolytic activity were observed for K562 [PD-L1 (52%), CTLA-4 (5%), CTLA-4 + PD-1 (7%) and PD-1 (7). A gentle increase in tumor cell lysis was observed in JX12T tumor cells for PD-1 (3%), CTLA-4 (2) PD-1 + CTLA-4 (5) combo and PD-L1 (8) However, checkpoint blocking did not increase the cytotoxicity of MGMT modified γδ T cells against JX22T and JX59T.

Our observations that glioblastoma tumor cells do not express significant levels of PD-L1 are consistent with reports already published by Garg et al. [The preclinical efficacy of immuno-checkpoint monotherapy is that the corresponding biomarker- Based clinical prediction. Onco Immunology, 2016]. A study by Joseph et al. Found a high level of PD-L1 expression in tumor invasive bone marrow cells of glioblastoma tumors [Immunosuppressive tumor-invasive bone marrow cells have been shown to induce adaptive immune tolerance through PD-1 / PD-L1 mechanisms in glioblastomas Neuro-Oncology 9 (6), 796-806, 2017]. Because of the findings of the present inventors that in the glioma glioma tumor microenvironment the bone marrow / substrate compartment expresses a high level of PD-L1 and that GMP-prepared T cells / gamma delta T cells upregulate PD-1, In order to treat and achieve a high objective response rate, we propose a comprehensive treatment plan that includes systemic treatment with PD-1, PD-L1 blocking antibody and temozolomide, and topical injection of MGδT-expressing γδ T cells.

The patents and scientific literature referred to herein establish the knowledge available to those skilled in the art. All U. S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims, . It is also to be understood that none of the embodiments described herein is mutually exclusive and can be combined in various ways without departing from the scope of the invention encompassed by the appended claims.

Claims (21)

i. obtaining a population of isolated cytotoxic immune cells comprising γδ T-cells, natural killer (NK) cells, or any combination thereof, wherein the population of cytotoxic immune cells is resistant to one or more therapeutic agents An entirely modified, step;
ii. Administering to the patient an effective amount of at least one of the therapeutically effective amount of the genetically modified cytotoxic immune cell of step (i);
iii. Administering to the patient a population of genetically modified cytotoxic immune cells of step (i); And
iv. Comprising administering to the patient an effective amount of at least one immune checkpoint inhibitor.
A method of treating cancer in a patient in need thereof. The method according to claim 1, wherein the cancer is selected from glioma, glioblastoma, lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, renal cell carcinoma, small cell lung cancer. 3. The method of claim 2 wherein the cancer is a glioma, glioblastoma, or neuroblastoma. 2. The method of claim 1, wherein the isolated cytotoxic immune cells comprise gamma delta T-cells, NK cells, and optionally further comprise other immunocompetent cells. The method of claim 4 wherein separating the cytotoxic immune cell is an alkyl guanine transferase (AGT), P140KMGMT, O ⁶ methyl guanine DNA methyl transferase (MGMT), L22Y-DHFR, thymidine delay tree synthetase, dihydro folate reductase Enzyme, or multidrug resistance-1 protein (MDR1). 6. The method of claim 5, wherein the isolated cytotoxic immune cells are genetically modified to encode P140KMGMT. The method of claim 1, wherein the immune checkpoint inhibitor is selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 CGEN-15049, CHK 1 kinase, CHK2 kinase, A2aR, OX40, or B-7 family ligand. 8. The method of claim 7, wherein the checkpoint inhibitor targets PD-1, PDL1, PDL2 or CTLA-4. The method of claim 1, wherein the immune checkpoint inhibitor is selected from the group consisting of tremelimumum, anti-OX40, PD-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475, OPDIVO (R) Wherein the antibody is selected from the group consisting of BY55 monoclonal antibody, AMP224, BMS-936559, MPLDL3280A, MSB0010718C, YERVOY® / eicilimumab, and Pembrolizumab (KEYTRUDA®). 7. The composition of claim 1, wherein the therapeutic agent is an alkylating agent; Metabolic antagonists; DNA demethylating agents; Substituted nucleotides; Substituted nucleosides; Antitumor antibiotics; Plant-derived antineoplastic agents; Cisplatin; Carboplatin; Etoposide; Methotrexate (MTX); Trimethotrexate (TMTX); Temozolomide; Ralitriptycide; S- (4-nitrobenzyl) -6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); Nitroso urea; Cytarabine; Camptothecin; And therapeutic derivatives thereof. 2. The method of claim 1, wherein the isolated cytotoxic immune cells are selected from the group consisting of alkylating agents; Metabolic antagonists; DNA demethylating agents; Substituted nucleotides; Substituted nucleosides; Antitumor antibiotics; Plant-derived antineoplastic agents; Cisplatin; Carboplatin; Etoposide; Methotrexate (MTX); Trimethotrexate (TMTX); Temozolomide; Ralitriptycide; S- (4-nitrobenzyl) -6-thioinosine (NBMPR); 6-benzylguanidine (6-BG); Nitroso urea; Cytarabine; Camptothecin; ≪ / RTI > and therapeutic derivatives thereof. 12. The method of claim 11, wherein the two therapeutic agents are temozolomide and methotrexate. 12. The method of claim 11, wherein the isolated cytotoxic immune cells are genetically modified with a drug resistant gene alkylguanine transferase (AGT) and a dihydrofolate reductase. 3. The method of claim 1, wherein administration of genetically modified immune cells of step (iii) and administration of the checkpoint inhibitor of step (iv) occurs substantially simultaneously or sequentially. At least one checkpoint inhibitor, and
a separate population of cytotoxic immune cells comprising gamma delta T-cells, NK cells, or any combination thereof,
Wherein greater than about 50% of the population of cytotoxic immune cells express a polypeptide conferring resistance to a chemotherapeutic agent. 16. The composition of claim 15, wherein the isolated population of cytotoxic immune cells comprises about 50% to about 95% gamma delta T-cells and about 5% to about 25% NK cells. 16. The method of claim 15, wherein the checkpoint inhibitor targets PD-1, PDL1, PDL2 or CTLA-4. The method of claim 15, wherein the cytotoxic immune cell is an alkyl guanine transferase (AGT), P140KMGMT, O ⁶ methyl guanine DNA methyl transferase (MGMT), L22Y-DHFR, thymidine delay tree synthetase, dihydro folate reductase, Or multi-drug resistance-1 protein (MDR1). The method of claim 1, wherein the isolated cytotoxic immune cells are derived from human induced pluripotent stem cells (hiPSC). 20. The method of claim 19, wherein the isolated cytotoxic immune cells comprise gamma delta T cells. 21. The method of claim 20, wherein the isolated cytotoxic immune cells further comprise NK cells.

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